Baseline Susceptibility of Filarial Vector Culex quinquefasciatus (Diptera: Culicidae) to Five Insecticides with Different Modes of Action in Southeast of Iran.

Background
Culex quinquefasciatus (Diptera: Culicidae) is an important vector for many human diseases. The aim of this study was to evaluate the susceptibility level of larval and adult stages of Cu. quinquefasciatus to different groups of WHO recommended insecticides for vector control.


Methods
Larval stages of the Culex mosquitoes were collected from their natural habitats in Rafsanjan County at Kerman Province, southeast of Iran in 2016. Insecticide susceptibility status of adult female Cx. quinquefasciatus against DDT (4%), deltamethrin (0.05%), malathion 5%, and bendiocarb (0.1%) were determined using WHO standard insecticide susceptibility test. Additional test was carried out to determine the susceptibility status of larvae of Cx. quinquefasciatus to temephos. Bioassay data were analyzed by Probit program.


Results
Cx. quinquefasciatus adults showed resistance to all four groups of the tested insecticides according to the WHO criteria for resistance evaluation. The lethal concentrations for 50% mortality (LC50) and 90% mortality (LC90) of temephos against Cx. quinquefasciatus larvae were 0.18mg/l and 0.78mg/l, respectively. This finding also confirms resistance to temephos based on the WHO recommended instructions for resistance evaluation.


Conclusion
Resistance to all groups of the tested insecticides should be considered for future vector control investigations in the study area.

Wastewater and sewage system are important breeding places for Culex mosquitoes.
Constant exposure of Cx. quinquefasciatus to the high organic content of wastewater including detergents, different groups of insecticides, industrial pollutants, and oil compounds can lead to the development of resistance in mosquito larvae against insecticides and larvicides (9)(10)(11). In recent years, increasing level of resistance to various groups of insecticides has been a major barrier to the success of vector control programs. Many studies have reported high level of resistance in Cx. quinquefasciatus to many groups of insecticides (12)(13)(14). Culex quinquefasciatus is an important member of Cx. pipiens complex wildly distributed worldwide (2,5,15).
In Iran, the resistance status of Cx. pipiens complex against different groups of insecticides was indicated the development of resistance in the members of this species, including Cx. quinquefasciatus, during the past quarter-century: development of resistance to most of the organochlorine insecticides including DDT (Dichloro diphenyl trichloroethane) (6,11,(16)(17)(18)(19)(20). Resistance to pyrethroid insecticides such as lambda-cyhalothrin, deltamethrin, and cyfluthrin (6,16,17,19). Resistance to the carbamate insecticides propoxur and bendiocarb (11,16,17) and relative resistance to malathion organophosphates insecticides (11,16). Moreover, resistance of the larvae of Cx. pipiens complex to temephos has recently been reported for the first time in Iran (9).
There was no study on monitoring the susceptibility level of Cx. quinquefasciatus to insecticides in Rafsanjan County at Kerman Province, southeastern Iran. We aimed to determine the susceptibility status of Cx. quinquefasciatus against insecticides in this area.

Study area
This study was carried out in Rafsanjan County at Kerman Province, southeastern Iran. The county located at latitude 30°30'N and longitude 55°40'E, with a population of 300000 in 2015 (Fig. 1).

Bioassay procedure
Larvae of Cx. quinquefasciatus were collected from larval habitats in Rafsanjan County in 2016, and all sample were transferred to laboratory and reared at 27 °C and 65±5% relative humidity using a 12h light/ 12h dark photoperiod. Bioassay tests were carried out using WHO test kits on adult mosquitoes (21). The following diagnostic concentrations of insecticides were tested: DDT 4%, lambdacyhalothrin 0.05%, malathion 5%, and bendiocarb 0.1%. Tests were carried out on3 to 5-day-old unfed females. Batches of 25 females were exposed to insecticide-impregnated papers at different exposure times. Two replicates of 25 adult mosquitoes (3 to 5-day-old unfed females) were considered as controls with untreated papers for each different exposure time. The lethal time for 50% mortality and 90% mortality (LT50 and LT90) among the mosquitoes was calculated using log-probit software according to Finney's formula (22,23). Larvicide susceptibility tests were carried out on late 3 rd to early 4 th stage larvae to determine larval susceptibility to temephos using WHO standard kit (1.25, 6.25, 31.25 and 156.25 mg/l concentrations), according to WHO instructions (22,23). Each test consisted of four replicates with 25 larvae each in glass beakers containing 250ml of distilled water and the specified insecticide concentration. Two replicates of 25 untreated larvae were maintained as controls. After 24h exposure period, larval mortality was calculated the lethal concentrations for 50% mortality and 90% mortality (LC50 and LC90) were calculated by probit analysis (24). In both adult and larval susceptibility testing, mortality rate in the test samples was corrected using Abbott formula (25), when the mortality rate of control was between 5% and 20%.

Results
The mortality rate (MR) in adult Cx. quinquefasciatus mosquitoes exposed to four different groups of insecticide-impregnated pa-  (Table 1).
Culex quinquefasciatus is resistant to malathion and bendiocarb and candidate of resistance to lambda-cyhalothrin and DDT based on the criteria for insecticide resistance described by WHO ( Table 2). The mortality rate in the mosquitoes at one-hour exposure to the insecticides calculated after 24h recovery period has been summarized in Table 2. Malathion had a mortality rate of 80% (MR= 80%), bendiocarb 88%, lambda-cyhalothrin and DDT 90% each, the mortality rate of DDT was calculated after 4h exposure time instead of 1h ( Table 2). The regression line of different concentration is shown in Fig. 2.
The mortality rate of each concentration of temephos is shown in Table 3. The mortality rate of temephos ranged from 3% to 100%. Moreover, using Finney's method, we calculated LC50 and LC90 for Cx. quinquefasciatus that were 0.18 and 0.78 ppm, respectively ( Table 4). The regression line of the different concentrations of temephos is shown in Fig. 3. A= y-intercept, B= the slope of the line, SE= standard error, CI= confidence interval, x 2 = heterogeneity about the regression line, df= degree of freedom, P> 0.05= represents no heterogeneity in the population of tested mosquitos.

Discussion
The present study provides evidence of resistance to four different classes of insecticides according to the current WHO criteria for insecticide resistance evaluation.
The mortality rate was interpreted as follows: higher than 98% was considered as susceptible, less than 90% indicated resistance, and from 90% to 97% was defined as resistance candidate. For the resistance candidate category (90-97% mortality rate), additional investigation is needed for the confirmation of resistance (21 This finding can be useful in future vector control programs and investigations in order to prevent the development of resistance to both insecticides. Previous studies have also reported Cx. pipiens resistance to different classes of insecticides in different geographical regions of Iran, resistance to DDT, lambda-cyhalothrin, deltamethrin and cyfluthrin in Tehran, capital of Iran (6,11,20). Resistance to DDT in the North of Iran (18), resistance to DDT, lambda-cyhalothrin and propoxur in a dirofilariasis foci in the Northwest of Iran (18), resistance to DDT, propoxur, cyfluthrin and lambda-cyhalothrin and tolerance to deltamethrin in a malaria endemic area in the Southeastern part of Iran (17), and resistance to deltamethrin and DDT in the Northwestern part of Iran (19).
The use of pesticides in agricultural sector can lead to the development of resistance to insecticides in medically important vectors including Cx. quinquefasciatus (13,(26)(27)(28).
There is a growing concern over the development of multiple insecticide resistance mechanisms in medically important arthropods that is a major problem in vector control (15,(29)(30)(31). Over the last fifty years, resistance to insecticides has been a growing concern. Resistance of mosquitoes to DDT was first reported in 1949 (32,33). However, resistance to organophosphorus insecticides in Cx. quinquefasciatus was first reported in 1961 (34), and to date, there have been several reports on resistance to various classes of insecticides in Cx. quinquefasciatus. This species is now quite resistant to some insecticides such as DDT and Malathion such that it does not exhibit mortality at one-hour exposure and 24h recovery period (35).
In the present study, in addition to adult susceptibility test, susceptibility of the larvae of Cx. quinquefasciatus to Temephos was evaluated according to WHO standard method.
We observed mortality rate ranged between 3% and 100%. In another study, laboratory evaluation of the susceptibility of Anopheles stephensi larvae collected from Kazeroun, south of Iran and Cx. pipiens larvae collected from Tehran, capital of Iran to temephos insecticides was carried out. LC50 values of both species were the same that is similar to our findings. Mosquito larvae in all the three geographical regions mentioned above have become resistant to temephos (9). In other countries were reported resistance of adult and larval stages of Cx. quinquefasciatus to different groups of insecticides and larvicides. In a study conducted in Morocco, bioassay results showed that Cx. pipiens is resistant to temephos that is consistent with our results (36). In Kuala Lumpur (Malaysia), Cx. quinquefasciatus larvae were found to be highly resistant to Malathion that was similar to the adults (35). In Central Tunisia, resistance to temephos in Cx. quinquefasciatus larvae have been reported (37) Moreover, resistance of Cx. quinquefasciatus larvae to malathion, permethrin, and resmethrin has been reported in Florida (USA) (38).
The use of pesticides in agriculture could play a role in the development of resistance to insecticides as well as larvicides in Cx. quinquefasciatus in Rafsanjan County (9,11).
Owing to the emergence of Cx. quinquefasciatus resistance to different classes of insecticides and larvicides, using some biological control agents such as Bacillus thuringiensis (a Gram-positive, soil-dwelling bacterium) and Gambusia affinis (larvivorous fish) can provide an efficient control strategy (39)(40)(41). Moreover, use of natural products derived from some plants such as Bunium persicum and Zhumeria majdae that have no adverse effects on the environment and humans can be suitable and alternative control approach for larvae as well as adult Cx. quinquefasciatus mosquitos (42)(43)(44)(45)(46)

Conclusion
Resistance to all tested insecticides was found. The high resistance status observed in the study area may be due to irregular use of pesticides in agriculture led to the constant exposure of the mosquito species to organic chemicals and subsequent development of resistance to insecticides and larvicides in Cx. quinquefasciatus. Therefore, regular monitoring of resistance status by standard bioassay and other complementary methods is necessary for the success of future chemical control programs.